Determining the presence and concentration of bio-molecules and other chemicals in a fluid is important in many applications. For example, an instrument that can determine the concentration of one or more specific chemical targets in a gas or liquid containing various chemicals may have applications in medical diagnostics, high throughput drug development, environmental testing, defense and laboratory-based research. Such techniques are also important for biomolecular interaction analysis in which reaction kinetics (on and off rates), affinity, and specificity are determined, along with other important parameters.
A common strategy to detect a chemical target is to use an instrument with a capture molecule which binds to the target chemical of interest and a transducer that allows the user to observe the binding event. Preferably, the capture molecule preferentially or exclusively binds to the chemical target. In the case of bio-molecular targets, antibodies, aptamers and polymers are used as capture molecules.
Optical transduction of binding events is a common detection method. To optically observe a binding event between a capture molecule and a target, various spectrometric techniques can be employed. These techniques may require that capture molecules be labeled with a transducer or tag, such as a fluorescent molecule for fluorescence spectroscopy or a Raman tag for Raman spectroscopy. A technique used for medical diagnostics is enzyme linked immunosorbant assay (ELISA) that utilizes fluorescently-labeled antibodies to detect various target chemicals, including bio-molecules, in human biological fluids to detect disease.
Labeled assays may be disadvantageous because labeled capture molecules may have adverse effects on assay results due to steric hindrances. Assays comprising labeled capture molecules are also not compatible with real-time testing. Labeling capture molecules also increases device complexity and cost.
Label-free assays, which do not require the addition of a labeled capture molecule, are advantageous because the target chemical is not sterically hindered from binding to the capture molecule by a label. Label-free assays may also measure binding events in real time, which improves the performance and sensitivity of the assay. Label-free assays can also be used for biomolecular interaction analysis as they provide real time data.
Metal nanoparticles, between 1 nm and 1000 nm in various dimensions, may be used as transducers in diagnostic assays. Some nanoparticle based diagnostic assays are ‘label-free’. Metal nanoparticle transducers can be used to monitor binding events in real time without additional labels through a phenomenon known as localized surface plasmon resonance (LSPR).
LSPR is a phenomenon associated with noble metal nanoparticles that creates sharp spectral absorbance and scattering peaks and produces strong electromagnetic near-field enhancements. These spectral peaks can be monitored using absorbance spectroscopy. The spectral peak changes with refractive index changes in the immediate vicinity of the nanoparticle surface. When chemical targets are bound near the surface of a metal nanoparticle, a shift in the spectral peak occurs due to changes in the local refractive index. This can be used to determine the concentration of a specific target in a complex medium.
LSPR sensors operate through the immobilization of metal nanoparticles onto a flat surface. The nanoparticles are functionalized with specific capture molecules, which may be an antibody. The sample fluid of interest is flowed over the top of the metal nanoparticles, the target chemicals of interest bind to their respective capture molecules, and the overall spectral peak of the sensor shifts according to the concentration of the chemical target on the capture molecules. In order to measure this shift, reflectance absorbance spectroscopy may be employed. Quantification is possible through comparing results to a previously-developed standard curve.
However, LSPR sensors suffer from low sensitivity and inadequate detection limits for a number of reasons.
LSPR sensors with nanoparticles on planar surfaces operate by flowing the sample longitudinally over the surface. In order for the sensor to determine the target concentration with the highest sensitivity and accuracy, the sensor must reach chemical equilibrium. Equilibrium occurs when the maximum fraction of capture molecule binding sites are occupied by chemical targets on the sensor surface, resulting in the largest sensor response in a reaction-limited assay. Lengthy incubation times are required to reach equilibrium.
Long incubation times are not suitable for many applications including point-of-care diagnostics. Long incubation times may be problematic for types of planar sensors other than LSPR sensors.
Reflectance LSPR signals from nanoparticles on a planar surface are also weak, leading to poor signal to noise ratios and poor detection limits. This may be addressed by using nanostructured surfaces to increase the surface area and nanoparticle density, resulting in a larger LSPR signal. However, this has the negative effect of increasing the time it takes to reach equilibrium and obtain the highest fraction of surface coverage since the number of surface sites is greatly increased. Essentially this improves signal to noise ratio but worsens the time to reach equilibrium, and overall does not greatly improve sensor performance. Moreover, these techniques rely on reflection measurement systems because the materials used are opaque at LSPR wavelengths and will not allow for transmission measurements.